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Candidatus Phytoplasma Mali’ in Different Populations of Cacopsylla Melanoneura in Italy

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Candidatus Phytoplasma Mali’ in Different Populations of Cacopsylla Melanoneura in Italy View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Archivio istituzionale della ricerca - Fondazione Edmund Mach Bulletin of Insectology 63 (1): 59-63, 2010 ISSN 1721-8861 Detection of ‘Candidatus Phytoplasma mali’ in different populations of Cacopsylla melanoneura in Italy 1 1 1 1 1 Valeria MALAGNINI , Federico PEDRAZZOLI , Valeria GUALANDRI , Rosaly ZASSO , Elisa BOZZA , Federica 1 2 2 1 FIAMINGO , Alberto POZZEBON , Nicola MORI , Claudio IORIATTI 1Centro Trasferimento Tecnologico, FEM- IASMA, San Michele all’Adige, Trento, Italy 2Department of Environmental Agronomy and Crop Science, University of Padua, Legnaro, Padova, Italy Abstract Cacospylla melanoneura Förster (Hemiptera Psyllidae) is one of the vectors of ‘Candidatus Phytoplasma mali’, which is the causal agent of apple proliferation (AP) disease. In 2006 and 2007, overwintering adult psyllids were collected from different host plants (apple, hawthorn and conifers) in different localities to assess the natural infection of C. melanoneura. AP phytoplasma was detected in insects through the use of PCR amplification with specific primers (AP3/AP4). Eleven percent of the psyllids collected from apple in the Trentino region were infected with AP phytoplasma, as compared with 18.63% of the psyllids collected from apple in the Aosta Valley and none of the psyllids collected from apple in the Veneto region. The percentage of AP-positive overwintering adults was higher in the Aosta Valley than in the Trentino region. Furthermore, considering the level of AP pres- ence in the monitored orchards, a positive correlation between the infection rates in the insects and the percentage of symptomatic plants was observed. Regarding psyllids collected from hawthorn, only few individuals tested positive in Trentino populations, while higher infection levels were found out in the Aosta Valley. Interesting percentages of positive psyllids resulted also in the insects collected from conifers (10.5%). Data obtained in this work demonstrate therefore that ‘Ca. P. mali’ may overwinter in the body of C. melanoneura and that there are differences in the infection proportion among populations. Key words: apple proliferation, phytoplasma, psyllids, Cacopsylla melanoneura, host plants. Introduction emerged with C. melanoneura, with only one successful transmission out of 278 test plants (transmission rate of The agronomic importance of the Hemiptera genus Ca- 0.36%) (Mattedi et al., 2008). copsylla is linked to the role played by several species Despite the incidence of ‘Ca. P. mali’-infected apple as vectors of phytoplasma-associated diseases. Apple plants in all the apple growing areas of Trentino, the proliferation (AP) disease is one of the most severe abundance of both species is dramatically decreasing in problems in Italian apple (Malus domestica Borkh.) or- the last years. Interesting population levels are only pre- chards. In northeastern Italy, symptoms of AP have sent for C. melanoneura in Valsugana, while C. picta been observed since the 1950s (Rui, 1950), but the dis- density has drastically dropped down in the whole re- ease has only recently become widespread, particularly gion and therefore this study focuses on the role of the in Trentino (northeastern Italy), where it has struck the first species in AP epidemiology. cultivars Golden Delicious, Florina and Renetta Canada The biology of C. melanoneura in the apple orchards (Vindimian and Delaiti, 1996). AP causes important of Trentino has been studied in detail (Mattedi et al., economic losses, as infected trees produce small fruits 2007; 2008). Overwintering adults migrate into apple with poor flavour. orchards between the end of January and February. The aetiological agent, ‘Candidatus Phytoplasma Oviposition begins between the end of February and the mali’ (Seemüller and Schneider, 2004), is transmitted beginning of March and this activity lasts about 30-40 by two psyllids, with different efficiencies: Cacopsylla days. The first nymphs appear at the end of March and picta (Förster) is the main vector in north-eastern Italy the new generation of adults emerges at the end of (Carraro et al., 2001b) and C. melanoneura (Förster) in April. As the adults develop, they migrate to other shel- north-western Italy (Tedeschi et al., 2002). Studies con- ter plants and disappear from the orchard before the end ducted in Germany confirm the role of C. picta in the of June. epidemiology of AP disease, excluding the latter species During the same period, C. melanoneura can also be (Jarausch B. et al., 2004; Mayer et al., 2009). Both spe- found on hawthorn (Crataegus monogyna Jacq. and C. cies are present in Trentino, but with different distribu- oxyacantha L.) (Ossiannilsson, 1992), on which high tions: C. melanoneura is ubiquist and shows the highest population densities have been reported even in some population densities in bottom valley environments locations of Trentino (Pedrazzoli et al., 2005). Recently, (Adige Valley and Valsugana); the presence of C. picta experiments conducted in north-western Italy found that is limited to hilly areas, such as Non and Sole Valley C. melanoneura individuals collected from hawthorn (Mattedi et al., 2008). Transmission experiments with carried AP-group phytoplasmas (Tedeschi et al., 2005; the two psyllids were therefore conducted also in this 2009). region over a six-years period (Mattedi et al., 2008). Besides hawthorn and apple, C. melanoneura has also Owing to these trials, the role of C. picta was confirmed been found on other plants, particularly on conifers such (transmission rate of 4.1%), while contradictory results as Picea abies (L.) H. Karst., Pinus sylvestris L., P. mugo Turra, on which the new generation of adults is Molecular analyses thought to overwinter (Conci et al., 1992; Ossiannils- Individual specimens were frozen, lyophilized, ho- son, 1992; Lauterer, 1999). P. abies is the main forest mogenized, and then stored at -80 °C until their DNA species of Trentino region, covering more than one third could be extracted. Total genomic DNA was extracted of the total forest area (137203 ha out of 407500 ha) from the samples following the CTAB (cetyltrimethyl- (INFC, 2005). To date, several studies have been con- ammonium bromide) method (Doyle and Doyle, 1990). ducted on the overwintering habits of this species in To assess the presence of ‘Ca. P. mali’, all of the col- Trentino (Pedrazzoli et al., 2005; Mattedi et al., 2007). lected insects were subjected to PCR amplification of a A regular presence has been found on Norway spruce non-ribosomal DNA region of 162 bp with the specific and pine in only a few high-altitude locations (Pedraz- primers AP3/AP4 (Jarausch et al., 1994). The PCR as- zoli et al., 2005). says were performed with 100-150 ng DNA, 375 nM of The different roles assigned to C. melanoneura by the each primer and the 2x Go Taq® Green Master Mix studies conducted to date suggest the existence of dif- (Promega, Madison, USA), in a final reaction volume of ferent populations, characterized by a different level of 20 µl. The PCR parameters were as follows: an initial infectivity. To examine these differences thoroughly, denaturation of 2 min at 95 °C and 40 cycles with 30 sec molecular analyses were performed on overwintered C. at 95 °C, 30 sec at 57 °C, 30 sec at 72 °C and a final ex- melanoneura collected from different plant species (ap- tension of 5 min at 72 °C. Amplifications were per- ple, hawthorn and conifers) in several areas of Italy. formed in a Gene Amp® PCR System 9700 (Applied Hawthorn hedgerows are a common component of the Biosystems, Foster City, CA, USA). The total DNA ex- rural landscape of Trentino region, so that often they are tracted from infected insects and a reaction blank (Milli- close to apple orchards. The knowledge of the infectiv- Q water) were included in the experiments as controls. ity of C. melanoneura collected from hawthorn plants PCR products were analyzed by electrophoresis on 1.5% could be informative about alternative inoculum w/v agarose gels stained with SYBR® Safe DNA gel sources. stain (Invitrogen, Carlsbad, CA, USA) and visualized In the present work, we report the results of these in- under UV light with the Gel Doc XR System molecular vestigations, underlining the correlation between the in- imager (BIO-RAD, Hercules, CA, USA; figure 1). fection level in the different plants and in the insects. Data analysis The correlation between the proportion of infected in- Materials and methods sects and the presence of symptomatic plants in apple or- chards was analysed applying the median test for the cor- Insect collection relation between two variables proposed by Blomqvist The research was conducted in the Trentino and Ve- (1950). As there is not an official classification of the neto regions (northeastern Italy) and in the Aosta Valley level of infected orchards, we did an arbitrary classifica- (northwestern Italy) between 2005 and 2007, as reported tion dividing apple orchards in four classes: low (with a in table 1. Overwintering adults of C. melanoneura were percentage of symptomatic plants ranging from 0 to 10), collected from apple trees and hawthorn bushes at the middle (from 10% to 30% of symptomatic plants), high end of March and from conifers during December and (from 30% to 50%) and very high (more than 50%). This January, using the beat tray method. The specimens analysis was not carried out for the other plant species, as were identified using Ossiannilsson’s keys (1992). no symptoms were detected on hawthorn and conifers. Table 1. Samples of C. melanoneura populations collected from different host plants in different locations. The to- tal number of psyllids analysed, the proportion of AP-infected samples and infection level in the sampled orchards are given. Insects Infection level in the Host plant Locality Location infected/tot. percentage sampled orchards Apple Borgo (Trentino) 46°3'N 11°29'E 11/96 11.46% very high " Oltrecastello (Trentino) 46°4'N 11°9'E 27/113 23.87% high " S.
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